Replication defective herpes simplex virus comprising heterologous inserts

ABSTRACT

Constructs for the delivery of sequences of interest to cells include a herpes virus latency active promoter (LAP) of the latency associated transcript (LAT) region. An internal ribosome entry site (IRES) is located downstream of the LAP, with a nucleotide sequence of interest downstream of the IRES. Stable, long-term expression including export of mRNA to the cytoplasm and translation of the encoded polypeptide, is found in neuronal and non-neuronal cells.

The present invention relates to constructs for delivery of sequences ofinterest to cells of an individual, for instance using recombinantviruses. This can have a therapeutic aim: and examples of the constructsand cells containing them can be useful also, for example, in productionof a polypeptide which can then be used as desired (e.g. as animmunogen). By employing a latency active promoter of a latencyassociated transcript (LAT) region of a herpes simplex virus, long-term,high-level expression of a reporter sequence can be achieved.

It is often desirable to deliver exogenous DNA to cells in order toprovide a missing gene or to help correct abnormal cellular behaviour.The present invention is not generally concerned with any difficultiesthat may be associated with delivery of nucleic acid to cells.

Many viruses have evolved to deliver nucleic acid into the nucleus ofthe cell, where it can be expressed. Certain viruses have beengenetically engineered to carry a gene to be delivered, and can deliverit to host cells such-as those the virus normally infects. Gene deliveryvectors have also been based on attenuated or genetically disabledvirus.

A number of genetically engineered viruses have been used to deliverforeign genes to cells both in vitro and in vivo. For certain purposesit is desirable for the gene to be stably expressed, producingbiologically active amounts of its product on a long term basis. Thisremains a problem in a number of contexts.

Herpes Simplex Virus (HSV) is a ubiquitous pathogen of man which iscapable of acutely infecting many cell types, and can persist long termin the sensory neurons of the host's dorsal root ganglia. This state,described as viral latency, is characterised by the persistence of theviral genome in the nucleus of the neuron without any detectableproduction of viral proteins, or interference with normal cellularmetabolism. HSV's ability to establish latency in neurons makes it anattractive-candidate as a gene delivery vector for the nervous system.

Though the virus does not produce any detectable protein product duringlatency, there is continuing RNA transcription. This latency associatedtranscription comes from a single region of the viral genome (thelatency associated transcript or LAT region) and is driven by thelatency active promoter (LAP). The TATA box and basal transcriptionalregulatory sequences, which constitute the core LAT promoter, resideapproximately 700 bp upstream of the 2 kb major LAT.

HSV-1 is considered a good candidate vector for CNS gene therapy becauseit is able to establish life-long latent infections in human sensoryneurons. During latency, the viral genome appears to be maintainedepisomally, and hence there appears to be no danger of insertionalmutagenesis or inactivation of host genes. Furthermore, there is nodetectable production of viral proteins during latency, and no evidencethat the latent state interferes with the normal metabolism of the hostcell.

Viral gene expression during latency appears to be limited to 2 or 3nuclear RNA species which accumulate to high levels in sensory neuronsharbouring latent virus (the LATs) (3). These transcripts are driven bya complex promoter region (LAP) (4). A functional-LAT region is notessential for the establishment of latency, and viruses whose LAT regionhas been deleted can still establish latency (5), and in some cases,express LATs (6). In fact it seems that the LATs may be involved inreactivation, as LAT negative viruses do not reactivate efficiently (7).Latency can be established in the absence of any viral gene expression,and seems to be a default pathway for the virus when it enters a cellwhere productive infection is not possible.

Studies using diverse promoters to drive expression of a reporter gene(usually β-galactosidase) in animal models, have shown transientreporter gene expression, but that this is not long-lived. This has ledto much work trying to define what elements of the LAT region andpromoter (LAP) are involved in maintaining its long-term transcriptionalactivity. Further work has been done to try to utilise the LAP and othersequence elements in the LAT region to facilitate long-term expressionof reporter genes.

A rabbit β-globin gene inserted downstream of the TATA box of the LAPmade β-globin RNA during latent infection, but at lower levels than LATin wild type infection (8). When the experiments were repeated using theendogenous LAT promoters to drive β-galactosidase or nerve growth factorgenes, no RNA could be detected by in situ hybridisation (9).

The same group have since used a recombinant defective virus with aMoloney Murine Leukaemia Virus (MMLV) LTR, Lac Z construct inserted intoICP4, and a deletion of the 5′ part of LAT, and demonstratedβ-galactosidase expression in sensory ganglia (10). Gene expression wasalso assessed in motor neurons of the hypoglossal nucleus, where therewas abundant transient expression. The results were taken to indicatethat the MMLV LTR did not remain active during latency in motor neurons.When the MMLV LTR is moved away from the viral repeats (where it is nearto endogenous LAT sequences) and inserted into the gC locus, it was notlonger able to produce long-term gene expression. If the region upstreamof the LAP is also inserted into the gC locus upstream of the LTR, thisvirus is capable of producing gene expression in sensory neurons. TheLAT sequences did not have similar facilitating effects when insertedupstream of the murine metallothionein promoter (11).

Other groups have also used the LAT promoter in vectors. Wolfe et alused a recombinant virus with the β-glucoronidsase (GUSB) gene insertedinto a LAT deletion downstream of the LAP in an attempt to correct thedeficiency in GUSB deficient mice (12). A corneal infection route wasused, and, although there was no phenotypic improvement in the conditionof the mice, they were able to detect some GUSB positive cells as muchas 18 weeks post inoculation. Miyanohara et al have used a variety ofHSV-1 vectors to attempt to deliver genes to the liver of mice (13).Using a LAT promoter to drive HbsAg or canine factor IX, they saw lowlevels of protein in the serum for about 3 weeks after direct injectionof the vector into the liver.

These studies suggest that the LAT promoter may be able to produceprolonged, albeit low level, expression of foreign genes in the CNS andeven in non-neuronal cells.

Goins et al. J. Virol. 68: 2239-2252 (1994) and WO96/27672 postulatedthe presence of a second latency-active promoter, tested for byexperiments involving transient gene expression, located in the HSV1U_(L) flanking repeats.

Specification WO 96/27672 (published later than the priority dateclaimed for the present application) (Glorioso & Fink) concerns thestructure of a herpesvirus promoter for transcription of a non-herpesgene in a cell latently infected with a herpes virus, e.g. peripheralneurons and cranial nerve ganglia.

HSV-1 based vectors have also been constructed using lytic cycle HSVpromoters (gC (14), IE110 (15)), strong non-specific promoters (CMV IEMMLV LTR) and neuron specific promoters (NSE (16)). On the whole, thesestudies show only transient gene expression in both peripheral and CNSneurons. As discussed above, the MMLV LTR can give long-term expression,but only when inserted close to LAT elements.

The present inventors now show experimentally that by inserting areporter gene, preceded by an internal ribosomal entry site (to allowefficient translation), downstream of the LAP in the LAT region, avector that gives stable, high level reporter gene expression duringviral latency can be produced.

In initial attempts to construct HSV vectors which would give long-termgene expression the present inventors decided to use a murine RNApolymerase I (RNA pol I) promoter reporter gene construct inserted intothe LAT region. RNA pol I is responsible for the transcription ofribosomal RNAs, and is active in all cell types. Native RNA pol Itranscripts are not capped, and are not recognised by the translationalapparatus of the cell, but, by inserting an internal ribosomal entrysite (IRES) sequence immediately upstream of the reporter gene, it ispossible to get efficient translation from such a transcript (17). Byinserting this construct into the LAT region of HSV, which is known tobe transcriptionally active during latency, the present inventors hopedto show RNA pol I activity during latent infection.

The experimental work is described in detail below. Unexpected resultswere obtained. No activity of the RNA pol I was detected during latencywhen a reporter construct was inserted into a HpaI deletion in the LATregion in the opposite orientation to the direction of LATtranscription. However, anti-sense transcripts of the reporter genesequence were detected in the cytoplasm, showing that the LAT region hadbeen modified for LAP activity to drive during latency expression oftranscripts which were exported to the cytoplasm. (Lachmann, Brown andEfstathiou (1996), J. Gen. Virol. 77: 2575-2582). An enzyme reportergene construct inserted into the same site for expression in the correctorientation gave efficient, long-term expression of enzyme activity.

According to a first aspect of the present invention there is provided anucleic acid construct comprising (i) a portion of the latencyassociated transcript (LAT) region of a herpes simplex virus (HSV)genome, which portion comprises a latency active promoter (LAP), (ii) aninternal ribosomal entry site (IRES) and (iii) a nucleotide sequenceheterologous to the herpes virus LAT region.

The heterologous nucleotide sequence may be said to be “operably linked”to the LAP and the IRES for expression of the sequence of amino acids.The term “operably linked” with respect to a nucleotide sequence. (suchas a coding sequence) and a promoter means that the nucleotide sequenceis positioned or disposed in the nucleic acid construct relative to thepromoter suitably for transcription of the nucleotide sequence to beunder the control of the promoter. With respect to a nucleotide sequenceand an IRES, the term “operably linked” means that the nucleotidesequence is positioned or disposed in the nucleic acid constructrelative to the IRES suitably for the IRES to perform its function, i.e.for translation of an mRNA transcript of the sequence to be stimulatedor enhanced (i.e. compared with an equivalent construct lacking an IRESoperably linked to the nucleotide sequence).

Elements of the LAT promoter important for long term gene expression arenot disrupted by insertion-of foreign DNA sequences downstream of theLAT transcription site. Transciption of heterologous sequences inconstructs according to the present invention may initiate from the LATtranscription start site, the transcribed RNA being exported to thecytoplasm of latently infected cells.

An IRES is an RNA sequence which facilitates ribosomal attachment andtranslation from an ATG methionine codon internal to an mRNA. Examplesknown per se include sequences encoded by picornaviruses. These havebeen divided into 3 groups; IRES sequences from entero and rhinoviruses, from cardio and apthoviruses and from hepatitis A virus. IRESsequences have also been described in other viruses such as hepatitis Cand recently in some non-viral organisms.

Mutants, variants and derivatives of naturally occurring IRES sequencesmay be employed in the present invention provided they retain theability to enhance translation.

The portion of the LAT comprising a LAP may be subject to mutation oralteration or one or more nucleotides, e.g. by insertion, addition,deletion or substitution to provide a mutant, variant or derivative of anaturally-occurring sequence. Those skilled in the art realise that itis possible to make changes to a nucleic acid molecule which either haveno effect on its function or will modulate the level of the activity ofinterest. Changes which do have an effect may modulate the level ofactivity in a manner which is useful, e.g. to increase the level ofexpression or any other desirable property. The present invention mustbe taken to extend to constructs comprising a mutant, variant orderivative of a LAT sequence, as long as expression is viable in asuitable host cell.

Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Molecular Cloning: aLaboratory Manual: 2nd edition, Sambrook et al., 1989, Cold SpringHarbor Laboratory Press and in Current Protocols in Molecular Biology,Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. Thedisclosures of Sambrook et al. and Ausubel et al. are incorporatedherein by reference (as are all references mentioned herein).

The IRES is generally placed downstream of the LAP, while theheterologous nucleotide sequence is placed downstream of the IRES. Theheterologous nucleotide sequence and IRES may be placed about 1.5 kbdownstream of the transcription start site of the LAT promoter.

The present inventors have found that such a construct may be used toprovide efficient, high-level and long-term expression especially wherethe IRES and coding sequence are inserted downstream of the LAP in placeof a HpaI restriction fragment of the LAT region. HpaI cuts afternucleotide 120,301 and before nucleotide 120,469 of the HSV-1 genome.The HSV-1 genome sequence is given in Perry and McGeoch (1988) J. Gen.Virol. 69:2831-2846. Attention is directed to the sequence informationprovided by Perry and McGeoch, incorporated herein by reference. Theboundaries of the LAT promoter are predicted to lie between coordinates117,010-120,301. They may lie between 118,439-120,301.

Thus, according to a further aspect of the present invention there isprovided a nucleic acid construct comprising (i) a portion of thelatency associated transcript (LAT) region of a herpes simplex virus(HSV) genome, which portion comprises a latency active promoter (LAP),and (ii) a heterologous nucleotide sequence inserted in the region of aHpaI restriction fragment of the LAT region downstream of the LAP. Theheterologous sequence may be inserted in place of the HpaI restrictionfragment, or in an adjacent or nearby site. Those skilled in the art canreadily determine, by experiments guided by the present disclosure andinvolving otherwise per se well known procedures, the variation which ispossible from the specific insertion described herein without abolishinglong-term gene expression.

Where the HSV genomic region employed is derived from HSV-2 rather thanHSV-1, the heterologous nucleotide sequence may be inserted at aroundthe same distance from the LAP transcription start site or in theequivalent genomic location in the HSV-2 sequence. The promoter regionsof HSV-1 and HSV-2 are very similar. Even though there is somedivergence in the sequence of the region in HSV-2 which is equivalent tothe HpaI insertion site, the person skilled in the art can readilydetermine, by experimentation guided by the present disclosure andinvolving otherwise per se well known procedures, the equivalentposition in relation to the LAP of HSV-2 for insertion of a heteroloussequence to give long-term gene expression as disclosed.

The term “heterologous” is used to refer to a nucleotide sequence whichis not normally or naturally found in the specified position within theLAT region. It may therefore be any sequence of nucleotides differentfrom the sequence of the fragment found naturally between the two HpaIrestriction sites in the LAT region of the herpes simplex virus, i.e.the HpaI restriction fragment. As used in relation to a herpes virus“heterologous” may be used to refer to a non-herpes viral sequence, or asequence not of the specific herpes virus in question. Possiblealternative terminology includes “foreign” or “exogenous”.

A heterologous nucleotide sequence may encode a sequence of amino acids,i.e. a peptide or a polypeptide.

Such a nucleotide sequence may be included in constructs of the presentinvention downstream of an IRES and/or in the region of the HpaIrestriction fragment, as discussed. Advantageously, the sequence ofamino acids is a protein such as a biologically functional protein whoseexpression from the construct in cells of an individual has atherapeutic effect.

Alternatively, the nucleotide sequence may on transcription produce aRNA molecule which is able to influence expression of another gene byantisense regulation. The use of anti-sense genes or partial genesequences to down-regulate gene expression is now well-established.Double-stranded DNA is placed under the control of a promoter in a“reverse orientation” such that transcription of the “anti-sense” strandof the DNA yields RNA which is complementary to normal mRNA transcribedfrom the “sense” strand of the target gene. The complementary anti-senseRNA sequence is thought then to bind with mRNA to form a duplex,inhibiting translation of the endogenous mRNA from the target gene intoprotein. Whether or not this is the actual mode of action is stilluncertain. However, it is established fact that the technique works.

Another possibility is that the nucleotide sequence on transcriptionproduces a ribozyme, able to cut nucleic acid at a specific site—andthus also useful in influencing gene expression. Background referencesfor ribozymes include Kashani-Sabet and Scanlon, 1995, Cancer GeneTherapy, 2(3): 213-223, and Mercola and Cohen, 1995, Cancer GeneTherapy, 2(1), 47-59.

In addition to the said heterologous nucleotide sequence, such as acoding sequence, the heterologous nucleotide sequence may comprise oneor more regulatory elements to enhance or improve transcription, and/or(for a coding sequence) translation.

In a further aspect, the present invention provides a nucleic acidconstruct including (a) a herpes viral LAT promoter, (b) locateddownstream of the promoter, a heterologous nucleotide sequence(polynucleotide), and (c) a further heterologous nucleotide sequenceencoding a polyA tail, located downstream of the nucleotide sequence(b). As discussed, a heterologous nucleotide sequence located downstreamof the promoter for transcription in the nucleus of a cell containingthe construct may encode any desired product, including a polypeptide,an antisense RNA and/or a ribozyme.

In preferred embodiments of various aspects of the present invention theheterologous nucleotide sequence comprises an internal ribosome entrysite (IRES) to enhance translation of the coding sequence.

Where a polynucleotide for transcription in a cell nucleus carrying aconstruct according to the present invention encodes an antisense orribozyme sequence, there is no need to provide a ribosomal entry site.

Constructs according to the present invention may be used for long-termexpression. For instance, expression may be for at least about 26 days,preferably at least about 82 days, more preferably at least about 140days, at least about 190 days, at least about 257 days, or at leastabout 307 days. We have observed long-term neuronal gene expression at257 days post-infection in the peripheral nervous system and up to 9-10months post infection in the central nervous system, including in thehypoglossal nucleus, facial nerve nucleus and cervical spinal cord. (SeeTable 1 and discussion in the experimental section below.)

Elements of the LAT promoter important for long term gene expression arenot disrupted by insertion of foreign DNA sequences downstream of theLAT transcription site. Transciption of heterologous sequences inconstructs according to the present invention may initiate from the LATtranscription start site, the transcribed RNA being exported to thecytoplasm-of latently infected cells.

Embodiments of the invention include the use of viruses such as forexample replication defective viruses to obtain long-term geneexpression in latently infected cells.

In this connection, the constructs may form part of a vector forintroduction into cells. The vector may be a plasmid. A viral vector maybe used such as an HSV vector or an HSV-derived vector, e.g. a mutantwhich is unable to initiate the cycle of productive infection and whichmay therefore be driven into the latent state or a mutant able toestablish latency in cell-types other than neurons. Replicationdefective and/or attenuated viruses may be preferred for certainpurposes.

A replication defective herpes virus may lack a functional form of oneor more of the regulatory proteins ICP0, ICP4, ICP22, ICP27 and ICP47,and may lacking additionally or alternatively the essential glycoproteinH (gH) gene, or a functional form thereof. Propagation of a replicationdefective virus requires a complementing cell line. Cell lines able tocomplement both ICP4 and ICP27 gene defects have been constructed bySamaniego et al. (J. Virol. (1995) 69: 5705-5715). CRI cells are able tosupply gH in trans.

A construct according to the present invention may for example beincluded in a replication defective virus which contains a deletion ofthe gH gene, and optionally also the ICP4 and ICP27 genes, which virusmay be propagated in a suitable complementing host cell.

Thus, according to a further aspect of the invention there is provided avirus or a viral particle comprising as part of its genetic make-up orgenome a construct as disclosed. This may be used to introduce theconstruct into a cell, e.g. a cell of an individual.

Cells comprising a construct according to the present invention areprovided as a further aspect of the invention, especially cells in whichthe construct is incorporated. Such cells may be in culture in vitro,and useful for study of expression etc., or may form part of a mammal,especially a non-human mammal such as a-primate or rodent such as amouse.

Transgenic animals comprising cells which comprise a construct asdisclosed also represent an aspect of the present invention, especiallynon-human mammals which may be used experimentally to investigateproperties of the construct and/or therapeutic potential of delivery ofany particular nucleotide sequence to cells of the body, such asneurons.

Methods which comprise introduction of a construct according to thepresent invention into a cell, e.g. by means of viral infection, arealso provided by the invention. This may be performed ex vivo (in vitro)or in vivo. A method according to the present invention may includecausing or allowing expression of a heterologous nucleotide sequence ina nucleic acid construct, for instance within a cell followingintroduction of the construct into the cell or an ancestor thereof. Acell containing a construct according to the invention, e.g. as a resultof introduction of the construct into the cell or an ancestor thereof,may be administered to a subject. Following such introduction, cells maybe cultured or maintained ex vivo and then delivered to a subject,either from which they were obtained (or from which an ancestor wasobtained) or a different subject. Where cells are to be used as animmunogen (for instance), they may be killed or inactivated prior toadministration.

Also provided is a method comprising administration of a compositioncomprising a construct as disclosed to an individual. The administrationmay be by infection with a viral vector which comprises the construct.Naked DNA delivery may be used.

Stereotactic injection of the therapeutic virus into the nervous systemas described by During et al. (ref. 2) is an accepted, efficient andwidely used procedure for introducing substances to, or biopsying from,specific regions of the CNS in both humans and animals.

A further method according to an aspect of the present inventionincludes administration of a herpes virus containing a construct asdisclosed.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to show benefit to a patient. Such benefit may beat least amelioration of at least one symptom. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. Prescription oftreatment, eg decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto active ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

For intravascular, cutaneous, subcutaneous, intramuscular, intraocularor intracranial injection, or direct injection into cerebrospinal fluid,injection into the biliary tree, or injection at the site of affliction,the active ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

The invention further provides a construct as disclosed, or a vectorsuch as a virus comprising such a construct, for use in a method oftreatment of-the human or animal body, and use of such a construct orsuch a vector in the manufacture of a medicament or composition for usein treatment of the human or animal body. Treatment may be byadministration as disclosed above.

Nucleic acid constructs and cells containing them are not just useful ina therapeutic context. It is generally useful to be able to expressnucleic acid stably within a host cell in vitro. The results reportedherein indicate that stable, long-term expression can be achieved usingconstructs of the invention. This may therefore be employed in theproduction of a product encoded by a nucleotide sequence of interest,heterologous to the LAT region of a herpes virus, which may if need berecovered from a host cell for subsequent use. Polypeptides, forinstance, are useful in raising antibodies in animals which may be usedin the generation of hybridomas for monoclonal antibody production, andselection of antibodies or other binding molecules on columns or bymeans of bacteriophage display. A polypeptide produced by expressionfrom a heterologous nucleotide sequence included in a constructaccording to the present invention may of course have a multitude ofother uses, depending on the nature of the polypeptide itself.

A cell according to the present invention may be used as an immunogen,for instance, after a period of culture or maintenance ex vivo to allowexpression of the heterologous polynucleotide, and optionally aftertreatment to kill or inactivate the cell.

Numerous other practical applications for constructs, vectors and cellsaccording to the present invention are suggested by the experimentalresults.

Nervous System Applications

Different Vector Systems

HSV establishes a natural latent state in sensory ganglia neurons thatcan last the life time of the individual. There is also experimentalevidence that defective HSV mutants, which have had certain of the genesrequired for a productive acute infection deleted, can also establishlatency in CNS neurons. Such a vector containing a construct accordingto the invention should result in long-term expression of these cells aswell.

The amplicon vector system consists of a plasmid containing the HSVorigin of replication and packaging signals, as well as a promoterreporter gene construct, which can be packaged in a herpes virion fordelivery (1). Such vectors produce good reporter gene expression incells in tissue culture. When used to try to target genes to neurons inanimal experiments, however, there has been less success. It has provedvery difficult to find promoter constructs that will allow high levelgene expression for long periods after infection. Insertion of aconstruct according to the present invention into an amplicon vector,maintains the LAT region sequences which give rise to long-term geneexpression in the native viral genome and should therefore producelong-term, high level expression of an inserted gene.

Similarly, the construct may be inserted into other viral vector systems(i.e. adenovirus, adeno-associated virus or retrovirus systems), or intoplasmids used in non-viral delivery systems (e.g. liposomes) to achievelong-term reporter gene expression in the nervous system.

Metabolic Diseases

There are many potential gene therapy applications within the nervoussystem. One of the main aims is to be able to complement various inbornerrors of metabolism which affect the CNS by delivery of a copy of themissing gene. For many such conditions (e.g. Gaucher's Disease) it hasbeen shown that the metabolic defects can be corrected by providingpharmacological amounts of the missing protein. Providing largequantities of such enzymes for pharmacotherapy is often not possible,and, where it is possible, extremely expensive. However, delivery usingthe present invention of the missing gene directly to the nervous systemcells with long-term expression of physiological levels of protein,provides for correction of the metabolic defect with a single treatment.Thus, the present invention may be used for treating a large number ofsuch inherited conditions such as neuronopathic Gaucher's disease andother lysosomal storage disorders, metachromatic leucodystrophy, G_(M2)gangliosidosis, Huntingdon's disease and so on.

There are other neurological diseases due to acquired metabolicabnormalities. In Parkinson's disease there is a loss of cells in thesubstantia nigra which leads to a deficiency of the neurotransmitterdopamine in the caudate nucleus. Patients can show a good therapeuticresponse to dopaminergic drugs, but if given orally large doses areneeded and the side effects often limit treatment. In this condition ithas been shown that providing a local source of the missing chemical inthe basal ganglia of the brain can lead to dramatic resolution ofsymptoms. By delivering the tyrosine hydroxylase gene (which codes theenzyme that makes dopamine) to the cells of the basal ganglia, it ispossible. to obtain symptomatic relief in experimental models of thedisease (2). Again the present invention provides a way of obtaininglong-term gene expression which may be useful for any of theseapplications. The approach may be usable for a number of otherdegenerative diseases of the nervous system, such as some of thedementias.

Neoplastic Diseases

Gene therapy also has applications in the treatment of cancer. There area few rare cancers and neoplastic syndromes that are due to inheritedgenetic defects (i.e. retinoblastoma, multiple endocrine neoplasia,neurofibromatosis). Some of these may be treated or even prevented bysupplying the missing gene by gene therapy. A number of these diseasescan affect the cells of the nervous system. Again vector constructsaccording to the invention may be useful in treatments.

All cancer involves the accumulation of a number of acquired geneticabnormalities which combine to lead to uncontrolled cell division andgrowth. As we learn more about neoplasia, it is becoming clear thatthere are a small number of genes which are very important in theaetiology of a large number of cancers. Tumour suppressor genes, such asp53, are good examples of genes which have become inactivated in manyneoplastic cell types. Gene therapy may be used to introduce afunctional copy of the mutated gene into the neoplastic cells, and henceto arrest their growth. Vectors as disclosed herein may be useful forsuch applications within the nervous system.

Another approach to gene therapy for cancer is to deliver a suicide genespecifically to the malignant cells which would produce a product whichcould, directly or indirectly, lead to their death. An example of such astrategy is to use the HSV thymidine kinase (TK) gene containing vectorsto specifically transduce malignant cells. The patient is then givenacyclovir, which is only metabolised to its active, toxic, metabolite incells which contain the HSV TK gene. With specific delivery of such asuicide gene to neoplastic cells, constructs of the invention may beused to produce gene expression in neural derived tissue.

Application Outside the Nervous System

Although the LAP is probably most active in neurons, experiments intissue culture have shown that it can be active in a wide range of othercell types. HSV can infect almost all cell types, though the--only knownsite of natural latency is in neurons. As noted above, however, it ispossible to make defective viruses which are not able to initiate thecycle of productive infection, and such a virus may be driven into thelatent state in other cell types.

Then, the LAT region may still be transcriptionally active, thoughpossibly at lower levels than in neurons. The advantage of using an IRES(as discussed) is that is allows very efficient translation of mRNA, andto get appreciable levels of protein product even from a low abundancetranscript. This holds for amplicon vectors as well as defective HSVvectors. Thus, HSV vectors containing a gene construct according to theinvention may be used for gene delivery to any of a wide range of celltypes, e.g. for tackling metabolic and neoplastic diseases, as describedabove.

Further aspects and embodiments of the present invention will beapparent to those skilled in the art, especially on consideration of thefollowing experimental exemplification which is provided by way ofillustration not limitation and with reference to the accompanyingfigures, wherein:

FIG. 1 shows the plasmid pMENA which contains the murine RNA polymerase1 promoter, an EMCV IRES and the neomycin resistance gene (ref. 17).

FIG. 2 shows a map of the LAT region encoded by HSV-1. A series ofrestriction fragments cloned into pbluescribe (Strategene) are shown.These plasmids are designated pSLAT1-pSLAT7. Introduction of foreign DNAsequences into the HSV genome was undertaken by deleting the HpaIfragment (nucleotide position 120,301-120,469 relative to the HSV-1genomic sequence) of pSLAT1. End repaired foreign DNA was inserted intothis locus. Linearized recombinant pSLAT1 was co-transfected with viralDNA and recombinant virus selected from the transfection progeny.

FIG. 3 shows a map to show the structure of viruses C3b, C3b⁺, C3bneoand C3bΔneo.

To make C3b and C3b⁺ a cassette of the CMV IE1 promoter drivingβ-galactosidase was blunt-end cloned into the HpaI deletion in pSLAT1(see FIG. 1). Clones containing the insert in each orientation wereselected. The recombinant viruses were made by co-transfection of verocells with PvuI linearised plasmid and HSV strain SC16 DNA. Transfectionprogeny was screened for β-galactosidase containing viruses, which wereplaque purified.

A similar strategy was used to make viruses C3bneo and C3bΔneo. Theinserts were prepared from pMENA and pΔMENA (FIG. 2) as XhoI NotIfragments. These were cloned into the HpaI deletion of pSLAT1.Recombinant viruses were made by co-transfecting vero cells withlinearised plasmid and C3b viral DNA. Recombinants no longer containingβ-galactosidase were plaque purified.

FIG. 4 shows plasmid pCA1.

This plasmid is based on the retroviral vector pBABE (ref: Nucleic AcidsResearch, 1990, 18, 3587). Into this a cassette containing theencephalomyocarditis virus IRES driving a β-galactosidase neomycinphosphotransferase fusion gene (β-geo) has been cloned. The IRES isderived from the plasmid pEMC2 (Ann Kaminski, Department ofBiochemistry, Cambridge).

FIG. 5 shows a map to show the structure of viruses LβA and LβB.

These viruses were constructed using the IRES-β-geo insert excised frompCA1 (FIG. 4) as an XbaI fragment. This was cloned into the HpaIdeletion of pSLAT1 (FIG. 1). Recombinant virus LβA was made byco-transfection of linearised plasmid with C3b⁺ viral DNA (FIG. 3). Therecombinants express limited β-galactosidase activity in acuteinfections in vitro, and virus was purified by selecting plaques thatdid not stain blue with the X-gal reagent. Southern blot analysis of thestructure of this virus shows that recombinant occurred within theβ-galactosidase-gene, and that no neoR sequences were present.

LβB was made by co-transfection with C3b viral DNA (FIG. 3) andrecombinant virus purified as above. This virus contains the completeIRESβgeo sequence.

FIG. 6 shows β-galactosidase expression in PNS from LβA (FIG. 6a) andLβB (FIG. 6b) infected mice. At each time point (days 4, 26, 82, and 190for LβA and days 5, 32 and 140) for LβB) cervical DRG CII--CIV weredissected from 5 to 6 mice and pooled for histochemical detection ofβ-galactosidase activity. After staining for 3 hours, the reaction wasstopped, and the ganglia clarified in glycerol prior to microscopicexamination and enumeration of the number of β-galactosidase expressingneurones.

All documents cited herein are incorporated by reference.

EXAMPLE 1 Vectors Containing RNA Polymerase I Constructs

A series of plasmids containing RNA pol I promoters driving expressionof the neomycin resistance gene, with an encephalomyocarditis virus IRESinserted immediately 5′ to the reporter gene (the pMENA series ofplasmids)-were obtained from Brian McStay (ref. 17) (FIG. 1). The MENAconstruct was excised as a Xho I Not I fragment, end-repaired and gelpurified. This insert was then blunt end cloned into the small Hpa Ideletion in pSLAT 1 (FIG. 2), a plasmid designed to allow homologousrecombination into the LAT region of HSV-1. Ampicillin resistantcolonies were picked, and restriction enzyme analysis confirmed thatclones containing the MENA insert in both orientations had beenobtained.

In order to avoid confusion with LAP activity, it was decided to insertthe MENA construct so that RNA pol I transcription would be in theopposite direction to LAT transcription (FIG. 3). The pSLAT1 clonecontaining the MENA insert in the correct orientation was prepared byCsCl ultra centrifugation, and linearised by digestion with Pvu I.

Viral DNA was prepared from Vero cells infected at multiplicity of 1pfu/cell with C3b, and HSV-1 based virus containing the Lac Z geneinserted into the LAT locus (FIG. 3). Cotransfections of Vero cells weredone using a CaCl₂ precipitation/DMSO shock protocol, and progeny viruswas harvested after 3 days, when plaques were beginning to form. Plaquepurification was performed under an agarose overlay containing the X-galsubstrate, which gives a blue precipitate where β-galactosidase ispresent, and white plaques were picked. After 3 rounds of plaquepurification, viral DNA was prepared, and Southern blot analysisperformed to confirm viral structure.

This virus, containing the MENA construct inserted into the LAT regionin the anti-sense direction to LAT transcription, is called C3bNeo. Afurther, similar virus was made using the same method to insert theΔMENA construct (containing a mutated, inactive RNA pol I promoter) intothe LAT region, C3bΔNeo (FIG. 3).

RNA pol I activity from these viruses was initially assessed duringacute infection of cells in tissue culture. Monolayers of BHK, Vero and3T3 cells were infected at high multiplicity, and lysates prepared atvarious time points for assay of neomycin phosphotransferase activity.The results showed that there is specific RNA pol I activity, mostactively in BHK cells, and that the IRES is able to allow efficienttranslation and production of protein product.

To investigate whether the C3bNeo virus exhibited any RNA pol I activityduring latency, 6 week old female Balb/C mice were infected with 2×10⁶plaque forming units (pfu) of C3bNeo by subcutaneous injection into theleft ear (20 μl injection volumes were used). Groups of 10 mice weresacrificed on day 30 and day 180 post infection. Left cervical dorsalroot ganglia 2 through 4 were dissected from each mouse, pooled andfixed in periodate lysine paraformaldehyde (PLP) fixative for 1 hour.They were then transferred to 50% ethanol prior to mounting in wax.

5 micron sections were cut and in situ hybridisation performed usingdigoxigenin labelled riboprobes for both sense and anti-sense (as acontrol) NeoR transcription. No neomycin phosphotransferase mRNA wasdetected, indicating that there was no detectable transcription from theRNA pol I promoter in viral latency. Surprisingly, however, we were ableto detect transcripts which were being made in the anti-sense directionto the MENA insert. These transcripts were localised to the cytoplasmand occurred at a frequency of 0.8 positive neurons per ganglionicsection examined.

It seemed most likely that these transcripts represented LAP activity,and that, by making a large insertion into the LAT region, we hadaltered LAT processing such that the transcripts were no longer retainedin the nucleus, but were exported to the cytoplasm. We confirmed thatthese cytoplasmic transcripts did contain LAT sequences by further ISHusing probes for the 2 kb LAT.

We have also demonstrated cytoplasmic LAT transcripts in latency withthe viruses C3b and C3b⁺ (FIG. 3). This shows that this phenomenon isnot specific to the MENA insert, but appears to happen whatever insertis placed into this small Hpa I deletion.

The nuclear LATs seen in wild type HSV latency are unlikely to representmRNA, as no translation can take place in the nucleus. It has beensuggested that they might represent a stable intron which has beenexcised from a larger mRNA, but conclusive evidence for such a largemRNA has not been found. Our insertion into the LAT region seems to haveled to the production of a LAT species which is distributed like a mRNA.

In effect, this is a virus where the LAP drives expression of an mRNAspecies which can be detected at high levels during latency. From this,on insertion of a reporter gene into this locus or other adjacent lociwithin major LAT, observation of long-term gene expression was expected.However, there are a number of stop codons between the TATA box of theLAP and the Hpa I deletion used for insertion of coding sequences, andit seemed unlikely that a reporter gene inserted directly into thislocus would be expressed.

EXAMPLE 2 Vectors Containing IRES-Reporter gene Constructs in the LATRegion

A plasmid containing an encephalomyocarditis virus IRES linked to a LacZ/Neo R fusion gene (termed β-Geo) (pCa1, FIG. 4) was obtained fromClare Abram (Dept of Pathology, Cambridge). The IRES-β-geo insert wascut out as an Xba I fragment, and blunt end cloned into the Hpa Ideletion of pSLAT 1. A clone containing the insert in the rightorientation, sense to LAT (pSLAT 1 β-Geo), was isolated, and DNAprepared by CsCl ultra centrifugation.

Two viruses have been made using this insertion plasmid. The first wasmade by co-transfection with viral DNA from C3b⁺ (FIG. 3). Therecombinant was plaque purified and its sequence analysed by Southernblot hybridisation. This showed that a the crossover had occurred duringrecombination within the Lac Z gene, and that the virus did not containany Neo R sequences. This virus is called LβA (FIG. 5).

A second virus was made by cotransfection of Vero cells with pSLAT 1β-Geo and C3b DNA. Again the recombinant virus was plaque purified andits structure confirmed by Southern blot. This virus is called LβB (FIG.5).

Both these viruses have a shared phenotype in acute infections of Verocells in tissue culture. If a suspension plaque assay is performed, andstaining for β-galactosidase performed, the plaques are predominantlywhite, but, if examined under the microscope, each plate will contain afew strongly positive blue cells, giving a unique speckled effect. Henceit seems that during acute infection of these non-neuronal cells, theLAP can be active, but only in a proportion of the cells. Whatconstitutes the switch for LAP activity is unclear.

LAP is supposed to show a degree of neuronal specificity, so LAPactivity from these viruses was studied during acute and latentinfections of mice.

11 female Balb/C mice, aged 5-6 weeks, were infected with 5×10⁶ pfu ofLβA in their left ears, and 5 mice with 5×10⁶ pfu C3b. 5 mice from eachgroup were sacrificed at day 5 post infection, and the remaining LβAmice were sacrificed at 26 days post infection. The cervical dorsal rootganglia 2-4 on the left were dissected out and these were pooled in the3 groups. Ganglia were fixed in 2% paraformaldehyde/0.2% glutaraldehydein PBS on ice for 1 hour. They were then stained for β-galactosidaseactivity using the X-gal reagent.

Ganglia were whole mounted under cover-slips, and examined under themicroscope. In the C3b⁺ infected mice, 3 of the ganglia were verystrongly positive with high level β-galacosidase expression, and it wasimpossible to count individual neurons. The day 5 LβA infected gangliashowed some positive staining, but this was not nearly as strong as thatseen with the virus containing the CMV IE promoter. There were onaverage 8.4 positive neurons per ganglion. By day 26 after LβAinfection, there was continuing β-galactosidase expression, and this wasseen at a higher level, and in more neurons, 20.3 per ganglion onaverage. Hence, activity of the native LAP is greater in latentlyinfected than acutely infected neurons, as would be expected, andefficient expression from the IRES-reporter gene construct persists intolatency.

At day 82 post-infection with LβA virus high level β-galactosidaseexpression was still seen in dorsal root ganglia at an average of 20.5blue neurones per ganglion examined. Examination at this time ofbrainstem and spinal cord showed abundant β-galactosidase expression inlarge numbers of neurones. Furthermore in ganglia and CNS tissue β-galexpression was detected in unidentified non-neuronal cell types—this isa novel finding. Ganglia containing a large number of neuronesexpressing β-galactosidase were readily detected 190 days postinfection.

Data are shown in FIG. 6a and Table 1. No obvious decrease in theaverage number of β-galactosidase expressing cells was evidentthroughout the time course of the experiments.

Following enumeration of β-gal positive neurones in whole mountedganglia sampled at 82 and 190 days post infection, tissues were paraffinembedded and sectioned on a microtome. This allowed an assessment of thenumber of blue neuronal profiles within ganglionic sections to be made.At 82 days after infection, 261 blue neuronal profiles were detected ina total of 271 ganglionic sections (an average of 0.96 “blue” neuronalprofiles per ganglionic section). From these data the conclusion is thatthere was no obvious decrease in the numbers of neurones expressingβ-galactosidase in ganglia removed from mice at between 82 and 190 dayspost-infection.

β-galactosidase expression in the peripheral nervous system of miceinfected with LβB has also been examined. FIG. 6b shows the numbers of“blue” neurones from whole mounted ganglia removed from animals atvarious times after infection. As observed for LβA, we were able todemonstrate long-term β-galactosidase expression withinlatently-infected sensory neurones up to 140 days after infection.However, through the course of this experiment it was noted that thenumbers of β-galactosidase positive cells per ganglion (Table 1), andthe intensity of staining of cells, appeared to be less with this virusthan as observed with LβA. Whole mounted ganglia, sampled from LβBinfected animals at 140 days after infection, were embedded in paraffin,and sections scored for “blue” neuronal profiles. 59 positive neuronalprofiles out of a total of 612 ganglionic sections were observed (anaverage of 0.1 “blue” neuronal profiles per ganglionic section)—a figureapproximately 10-fold lower than that obtained from LβA material sampledand analysed in a similar manner at 82 and 190 days post infection. Inorder to determine whether these observed differences between LβA andLβB were due to differences in the efficiencies of lacz during latency,in situ hybridisation (ISH) analyses were performed.

Cervical ganglia (CII-CIV) were dissected and pooled from mice latentlyinfected with LβA and LβB at various time points, and processed for ISH.ISH was performed using a digoxigenin labelled riboprobe which wasspecific for the detection of lacZ mRNA. The data from these experimentsare summarised in Table 1. At 82 days post-infection with LβA, 44 lacZRNA positive neuronal profiles were detected out of 167 ganglionicsections examined (an average of 0.26 lacZ positive neuronal profilesper ganglionic section), and at 190 days post infection, 42 lacZ RNApositive neuronal profiles were detected out of 162 ganglionic sectionsexamined (an average of 0.26 lacZ positive neuronal profiles perganglionic section). In each case the signal obtained was predominantlycytoplasmic, and the intensity of signal and mean number of lacZpositive neuronal profiles detected per section were similar at bothtime points. These data indicate the stable, continued transcription oflacZ specific RNA in sensory ganglia latently infected with LβA.

ISH analyses using a LAT specific probe also resulted in the detectionof transcripts within the cytoplasm of latently infected neurones, whichis in sharp contrast to the characteristic nuclear localisation of LATsobserved during latency with wild type viruses. The localisation of LATsand lacZ specific signals in the cytoplasm of neurones supports the viewthat hybrid transcripts are generated and transported to the cytoplasmof latently infected neurons. Interestingly, ISH detection of laczspecific RNA in ganglia latently-infected with LβB revealed a differentpattern of signal than that observed with LβA. Instead of the uniformcytoplasmic lacZ specific signal observed with LβA, animals latentlyinfected with LβB demonstrated a predominantly nuclear, punctate signal,with a cytoplasmic signal observed in only some of the more intenselystained cells. At 140 days post infection with LβB, ISH analysesrevealed 23 lacZ positive neuronal positive profiles out of 128ganglionic sections examined (an average of 0.18 neuronal lacZ positiveprofiles/ganglionic sections), and at 257 days post infection 102 lacZpositive profiles were observed in 288 ganglionic sections examined (anaverage of 0.36 lacZ positive neuronal profiles/ganglionic section). Ittherefore appears that LβB established transcriptionally active latencyat an efficiency comparable to that of LβA. However, despite the factthat similar numbers of latently infected neurones harbourtranscriptionally active LβA or LβB genomes, fewer neurones latentlyinfected with LβB scored positive for functional β-galactosidase geneexpression. The ISH data suggest that this may be a result of therelatively inefficient translocation of lacZ containing transcripts fromthe nucleus to the cytoplasm of neurones latently infected with LβBrather than failure of LAT promoter mediated transcription.

β-Galactosidase Expression in Neurones of the Central Nervous SystemFrom Mice Latently Infected with LβA

Using the mouse ear model it has previously been demonstrated that,following the resolution of acute phase infection, animals harbourlatent virus DNA in both brainstem and spinal cord tissue, as well asperipheral sensory ganglia (Efstathiou et al. (1986) J. Virol. 57:446-495). It is also well established that, using this model ofinfection, virus gains access to the brainstem via the facial nerve,which supplies motor fibres to the ear muscles (Hill et al. Prog. BrainRes. (1983) 59: 173-184).

We were, therefore, interested to determine whether latently infectedanimals would contain neurones expressing β-galactosidase at thesesites. To date our studies have focused on an examination of brainstemsand cervical spinal cords of mice latently infected with recombinantvirus LβA, and has involved analysis of: 3 mice sampled at 2-3 monthspost infection; 2 mice sampled at 4 months post infection; 1 mousesampled at 6 months post infection; and 11 mice sampled at 9-10 monthspost infection. As observed previously in our examination ofβ-galactosidase expression in latently infected sensory ganglia, therewas considerable mouse to mouse variation in the level oftranscriptionally active latency established. However, the anatomicaldistribution of β-galactosidase expression was maintained, andβ-galactosidase expressing neurones were consistently detected in thecervical spinal cord, and bilaterally within the facial nerve nuclei andhypoglossal nerve nuclei.

β-galactosidase expression was detected most frequently in the facialnerve nuclei (14 out of 17 mice examined), and ‘blue’ neurones wereoften observed throughout the nucleus (in up to 13 consecutive 60 μmsections), with some neurones showing tracking of β-galactosidase intothe dendritic tree and axon. The wide distribution of latently infected‘blue’ neurones within the facial nucleus would be indicative of virusspread within the nucleus during acute infection. β-galactosidasepositive neurones were detected in the hypoglossal nuclei in 8 of the 17animals examined. Latently infected ‘blue’ neurones were again commonlyobserved throughout the nucleus (in up to 15 consecutive 60 μmsections). Cervical spinal cord was not obtained from all mice butβ-galactosidase expression was observed in both anterior and posteriorhorn neurones in 6 out of 14 mice examined. We have observed occasionalβ-galactosidase expressing neurones in the region of the dorsal columnsensory nuclei within the posterior caudal medulla of some mice as wellas ‘blue’ axonal profiles traversing this area. These are likely torepresent axons projecting from latently infected sensory neuroneslocated in dorsal root ganglia.

The results obtained using embodiments of the present invention are insharp contrast to most other attempts to obtain long-term geneexpression from HSV based vectors, where gene expression has been at itsstrongest during the acute infection, and then tailed off duringestablishment of latency.

Examination of brainstem and spinal cord tissues of mice latentlyinfected with LβA revealed β-galactosidase positive neurones in a numberof distinct regions of the CNS at time points ranging from 72 to 307days after infection.

For construction of a long-term expression cassette which can be used inother vector systems, the LAP-IRES β-geo cassette is being cloned intoother regions of the virus, and into an amplicon vector. This is bycloning the Not-1 fragment which contains the LAP (from 118,439 to122,025 bp) and/or a HpaI fragment (nucleotides 117,010 to 120,301) fromthe Bam HI B fragment of HSV-1 into the NotI site of pcDNA3 (Invitrogen)to generate pcDNA3/LAT. The XbaI fragment of pCA-1 is then cloneddownstream of the LAP sequence at a position corresponding to HSVnucleotide 120,301 in pcDNA3/LAT. The LAP-IRES β-geo cassette can now beexcised as a NotI fragment and cloned into HSV amplicons or into otherviral loci, or used as a plasmid for naked DNA delivery.

REFERENCES

1. Spaete & Frenkel, Cell 30, 295-304 (1982).

2. During, et al., Science 266, 1399-403 (1994).

3. Feldman, Seminars in virology 5, 207-212 (1994).

4. Goins, et al., J Virol 68, 2239-52 (1994).

5. Deshmane, et al., Virology 196, 868-72 (1993).

6. Nicosia, et al., J Virol 67, 7276-83 (1993).

7. Block, et al., Virology 192, 618-30 (1993).

8. Dobson, et al., J Virol 63, 3844-51 (1989).

9. Margolis, et al., Virology 197, 585-92 (1993).

10. Dobson, et al., Neuron 5, 353-60 (1990).

11. Lokensgard, et al., J Virol 68, 7148-58 (1994).

12. Wolfe, et al., Nat Genet 1, 379-84 (1992).

13. Miyanohara, et al., New Biol 4, 238-46 (1992).

14. Fink, et al., Hum Gene There 3, 11-9 (1992).

15. Weir et al., PNAS USA 90, 9140-4 (1993).

16. Andersen, et al., Hum Gene There 3, 487-99 (1992).

17. Palmer, et al., Nucleic Acid Res 21, 3451-7 (1993).

TABLE 1 Detection of β-galactosidase expression by histochemicalstaining and by ISH for mRNA in the peripheral nervous system of miceinfected with LβA and LβB. Average number of ISH positive neuronal Dayspost blue neurones profiles per Virus infection per ganglion (range)ganglionic section LβA 4  8.4 (0-55) ND 26 17.l (0-53) ND 82 19.8 (0-55)0.26 (44/167) 190 14.7 (0-85) 0.26 (42/162) LβB 5  2.8 (0-12) ND 32 11.7(0-43) 0.36 (68/187) 140 14.4 (1-32) 0.18 (23/128) 257 ND 0.36 (102/288)

What is claimed is:
 1. A recombinant replication defective herpessimplex virus comprising a herpes virus latency active promoter (LAP),an internal ribosome entry site (IRES) operably linked to and downstreamof the LAP, and a non-herpes virus nucleotide sequence downstream of theIRES, whereby said herpes simplex virus is able to express saidnon-herpes virus nucleotide sequence.
 2. An isolated cell which has beeninfected by a recombinant herpes virus according to claim
 1. 3. A cellaccording to claim 2, wherein the cell is a nerve cell.
 4. Therecombinant herpes virus of claim 1, wherein said heterologousnucleotide sequence and said internal ribosome entry site are insertedabout 1.5 kb downstream of a transcription start site of the latencyactive prqmoter (LAP).
 5. The recombinant herpes virus of claim 1,wherein said heterologous nucleotide sequence and said internal ribosomeentry site are inserted about 700 base pairs downstream of a corelatency associated transcript (LAT) promoter, wherein said core LATpromoter comprises a TATA box and a basal transcriptional regulatorysequence.
 6. A method for expressing a heterologous nucleic acidsequence in a cell comprising: introducing a virus according to claim 1into a cell, wherein said introduction results in the cell expressingthe nucleic acid.
 7. A method according to claim 6, wherein the cell isa nerve cell.
 8. A method according to claim 6, wherein the cell is invivo.
 9. A method for expressing a nucleic acid in a subject, whichcomprises administering to the subject a recombinant herpes virusaccording to claim 1, thereby expressing the nucleic acid in thesubject.